Ultrasonic contrast agent with polymeric gas or air filled microballoons

ABSTRACT

Ultrasonic contrast compositions containing gas or air filled microballoons having a mean size in the range of 0.5 to 1000 microns bounded by a 50 to 500 nm thick biodegradable, interfacially deposited, synthetic polymer membrane which is deformable and resilient in an aqueous carrier liquid are described.

RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/033,435, filed 18 Mar.1993, abandoned, which, in turn, is a continuation of application Ser.No. 07/695,343, filed May 3, 1991, abandoned. Priority rights under 35U.S.C. §120 are hereby claimed under all prior related U.S.applications.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns air or gas filled microcapsules ormicroballoons enclosed by an organic polymer envelope which can bedispersed or suspended in aqueous media and used in this form for oral,rectal and urethral applications or for injection into living beings,for instance for the purpose of ultrasonic echography and other medicalapplications.

The invention also comprises a method for making said microballoons inthe dry state, the latter being instantly dispersible in an aqueousliquid carrier to give suspensions with improved properties overexisting similar products. Hence, suspensions of the microballoons in acarrier liquid ready for administration are also part of the invention.

2. Related Art

It is well known that microbodies or microglobules of air or a gas, e.g.microspheres like microbubbles or microballoons, suspended in a liquidare exceptionally efficient ultrasound reflectors for echography. Inthis disclosure the term of "microbubble" specifically designates air orgas microspheres in suspension in a carrier liquid which generallyresult from the introduction therein of air or a gas in divided form,the liquid preferably also containing surfactants or tensides to controlthe surface properties and the stability of the bubbles. In themicrobubbles, the gas to liquid interface essentially comprises looselybound molecules of the carrier liquid. The term of "microcapsule" or"microballoon" designates preferably air or gas bodies with a materialboundary or envelope of molecules other than that of the carrier liquid,i.e. a polymer membrane wall. Both microbubbles and microballoons areuseful as ultrasonic contrast agents. For instance injecting into thebloodstream of living bodies suspensions of gas microbubbles ormicroballoons (in the range of 0.5 to 10 μm) in a carrier liquid willstrongly reinforce ultrasonic echography imaging, thus aiding in thevisualization of internal organs. Imaging of vessels and internal organscan strongly help in medical diagnosis, for instance for the detectionof cardiovascular and other diseases.

The formation of suspensions of microbubbles in an injectable liquidcarrier suitable for echography can be produced by the release of a gasdissolved under pressure in this liquid, or by a chemical reactiongenerating gaseous products, or by admixing with the liquid soluble orinsoluble solids containing air or gas trapped or adsorbed therein.

For instance, in U.S. Pat. No. 4,446,442 (Schering), there are discloseda series of different techniques for producing suspensions of gasmicrobubbles in a sterilized injectable liquid carrier using (a) asolution of a tenside (surfactant) in a carrier liquid (aqueous) and (b)a solution of a viscosity enhancer as stabilizer. For generating thebubbles, the techniques disclosed there include forcing at high velocitya mixture of (a), (b) and air through a small aperture; or injecting (a)into (b) shortly before use together with a physiologically acceptablegas; or adding an acid to (a) and a carbonate to (b), both componentsbeing mixed together just before use and the acid reacting with thecarbonate to generate CO₂ bubbles; or adding an over-pressurized gas toa mixture of (a) and (b) under storage, said gas being released intomicrobubbles at the time when the mixture is used for injection.

One problem with microbubbles is that they are generally short-livedeven in the presence of stabilizers. Thus, in EP-A-131.540 (Schering),there is disclosed the preparation of microbubble suspensions in which astabilized injectable carrier liquid, e.g. a physiological aqueoussolution of salt, or a solution of a sugar like maltose, dextrose,lactose or galactose, is mixed with solid microparticles (in the 0.1 to1 μm range) of the same sugars containing entrapped air. In order todevelop the suspension of bubbles in the liquid carrier, both liquid andsolid components are agitated together under sterile conditions for afew seconds and, once made, the suspension must then be usedimmediately, i.e. it should be injected within 5-10 minutes forechographic measurements; indeed, because the bubbles are evanescent,the concentration thereof becomes too low for being practical after thatperiod.

Another problem with microbubbles for echography after injection issize. As commonly admitted, microbubbles of useful size for allowingeasy transfer through small blood vessels range from about 0.5 to 10 μm;with larger bubbles, there are risks of clots and consecutive emboly.For instance, in the bubble suspensions disclosed in U.S. Pat. No.4,446,442 (Schering) in which aqueous solutions of surfactants such aslecithin, esters and ethers of fatty acids and fatty alcohols withpolyoxyethylene and polyoxyethylated polyols like sorbitol, glycols andglycerol, cholesterol, or polyoxy-ethylene-polyoxypropylene polymers,are vigorously shaken with solutions of viscosity raising andstabilizing compounds such as mono- and polysaccharides (glucose,lactose, sucrose, dextran, sorbitol); polyols, e.g. glycerol,polyglycols; and polypeptides like proteins, gelatin, oxypolygelatin andplasma protein, only about 50% of the microbubbles are below 40-50 μmwhich makes such suspensions unsuitable in many echographic application.

In contrast, microcapsules or microballoons have been developed in anattempt to cure some or the foregoing deficiencies. As said before,while the microbubbles only have an immaterial or evanescent envelope,i.e. they are only surrounded by a wall of liquid whose surface tensionis being modified by the presence of a surfactant, the microballoons ormicrocapsules have a tangible envelope made of substantive materialother than the carrier itself, e.g. a polymeric membrane with definitemechanical strength. In other terms, they are microspheres of solidmaterial in which the air or gas is more or less tightly encapsulated.

For instance, U.S. Pat. No. 4,276,885 (Tickner et al.) discloses usingsurface membrane microcapsules containing a gas for enhancing ultrasonicimages, the membrane including a multiplicity of non-toxic andnon-antigenic organic molecules. In a disclosed embodiment, thesemicrobubbles have a gelatin membrane which resists coalescence and theirpreferred size is 5-10 μm. The membrane of these microbubbles is said tobe sufficiently stable for making echographic measurements; however itis also said that after a period of time the gas entrapped therein willdissolve in the blood-stream and the bubbles will gradually disappear,this being probably due to slow dissolution of the gelatin. Before use,the microcapsules are kept in gelatin solutions in which they arestorage stable, but the gelatin needs to be heated and melted to becomeliquid at the time the suspension is used for making injection.

Microspheres of improved storage stability although without gelatin aredisclosed in U.S. Pat. No. 4,718,433 (Feinstein). These microspheres aremade by sonication (5 to 30 KHz) of viscous protein solutions like 5%serum albumin and have diameters in the 2-20 μm range, mainly 2-4 μm.The microspheres are stabilized by denaturation of the membrane formingprotein after sonication, for instance by using heat or by chemicalmeans, e.g. by reaction with formaldehyde or glutaraldehyde. Theconcentration of stable microspheres obtained by this technique is saidto be about 8×10⁶ /ml in the 2-4 μm range, about 10⁶ /ml in the 4-5 μmrange and less than 5×10⁵ in the 5-6 μm range. The stability time ofthese microspheres is said to be 48 hrs or longer and they permitconvenient left heart imaging after intravenous injection. For instance,the sonicated albumin microbubbles when injected into a peripheral veinare capable of transpulmonary passage. This results in echocardiographicopacification of the left ventricle cavity as well as myocardialtissues.

Recently still further improved microballoons for injection ultrasonicechography have been reported in EP-A-324.938 (Widder). In this documentthere are disclosed high concentrations (more than 10⁸) or air-filledprotein-bounded microspheres of less than 10 μm which have life-times ofseveral months or more. Aqueous suspensions of these microballoons areproduced by ultrasonic cavitation of solutions of denaturable proteins,e.g. human serum albumin, which operation also leads to a degree offoaming of the membrane-forming protein and its subsequent hardening byheat. Other proteins such as hemoglobin and collagen are said to beconvenient also.

Still more recently M. A. Wheatley et al., Biomaterials 11 (1990),713-717, have reported the preparation of polymer-coated microspheres byionotropic gelation of alginate. The reference mentions severaltechniques to generate the microcapsules; in one case an alginatesolution was forced through a needle in an air jet which produced aspray of nascent air filled capsules which were hardened in a bath of1.2% aqueous CaCl₂. In a second case involving co-extrusion of gas andliquid, gas bubbles were introduced into nascent capsules by means of atriple-barelled head, i.e. air was injected into a central capillarytube while an alginate solution was forced through a larger tubearranged coaxially with the capillary tube, and sterile air was flownaround it through a mantle surrounding the second tube. Also in a thirdcase, gas was trapped in the alginate solution before spraying either byusing a homogeneizer or by sonication. The microballoons thus obtainedhad diameters in the range 30-100 μm, however still oversized for easilypassing through lung capillaries.

The high storage stability of the suspensions of microballoons disclosedin EP-A-324.938 enables them to be marketed as such, i.e. with theliquid carrier phase, which is a strong commercial asset sincepreparation before use is no longer necessary. However, the proteinmaterial used in this document may cause allergenic reactions withsensitive patients and, moreover, the extreme strength and stability ofthe membrane material has some drawbacks: for instance, because of theirrigidity, the membranes cannot sustain sudden pressure variations towhich the microspheres can be subjected, for instance during travelthrough the blood-stream, these variations of pressure being due toheart pulsations. Thus, under practical ultrasonic tests, a proportionof the microspheres will be ruptured which makes imaging reproducibilityawkward; also, these microballoons are not suitable for oral applicationas they will not resist the digestive enzymes present in thegastrointestinal tract. Moreover, it is known that microspheres withflexible walls are more echogenic than corresponding microspheres withrigid walls.

Furthermore, in the case of injections, excessive stability of thematerial forming the walls of the microspheres will slow down itsbiodegradation by the organism under test and may result intometabolization problems. Hence it is much preferable to develop pressuresustaining microballoons bounded by a soft and elastic membrane whichcan temporarily deform under variations of pressure and endowed withenhanced echogenicity; also it might be visualized that micro-balloonswith controllable biodegradability, for instance made of semi-permeablebiodegradable polymers with controlled micro-porosity for allowing slowpenetration of biological liquids, would be highly advantageous.

BRIEF SUMMARY OF THE INVENTION

These desirable features have now been achieved with the microballoonsof the present invention as defined in the claims Moreover, although thepresent microspheres can generally be made relatively short-lived, i.e.susceptible to biodegradation to cope with the foregoing metabolizationproblems by using selected types of polymers, this feature (which isactually controlled by the fabrication parameters) is not a commercialdrawback because either the microballoons can be stored and shipped dry,a condition in which they are stable indefinitely, or the membrane canbe made substantially impervious to the carrier liquid, degradationstarting to occur only after injection. In the first case, themicroballoons supplied in dry powder form are simply admixed with aproportion of an aqueous phase carrier before use, this proportion beingselected depending on the needs. Note that this is an additionaladvantage over the prior art products because the concentration can bechosen at will and initial values far exceeding the aforementioned 10⁸/ml, i.e. in the range 10⁵ to 10¹⁰, are readily accessible. It should benoted that the method of the invention (to be disclosed hereafter)enables to control porosity to a wide extent; hence microballoons with asubstantially impervious membrane can be made easily which are stable inthe form of suspensions in aqueous liquids and which can be marketed assuch also.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Microspheres with membranes of interfacially deposited polymers althoughin the state where they are filled with liquid, are well known in theart. They may normally result from the emulsification into droplets (thesize of which is controllable in function to the emulsificationparameters) of a first aqueous phase in an organic solution of polymerfollowed by dispersion of this emulsion into a second water phase andsubsequent evaporation of the organic solvent. During evaporation of thevolatile solvent, the polymer deposits interfacially at the dropletsboundary and forms a microporous membrane which efficiently bounds theencapsulated first aqueous phase from the surrounding second aqueousphase. This technique, although possible, is not preferred in thepresent invention.

Alternatively, one may emulsify with an emulsifier a hydrophobic phasein an aqueous phase (usually containing viscosity increasing agents asemulsion stabilizers) thus obtaining an oil-in-water type emulsion ofdroplets of the hydrophobic phase and thereafter adding thereto amembrane forming polymer dissolved in a volatile organic solvent notmiscible with the aqueous phase.

If the polymer is insoluble in the hydrophobic phase, it will depositinterfacially at the boundary between the droplets and the aqueousphase. Otherwise, evaporation of the volatile solvent will lead to theformation of said interfacially deposited membrane around the dropletsof the emulsified hydrophobic phase. Subsequent evaporation of theencapsulated volatile hydrophobic phase provides water filledmicrospheres surrounded by interfacially deposited polymer membranes.This technique which is advantageously used in the present invention isdisclosed by K. Uno et al. in J. Microencapsulation 1 (1984), 3-8 and K.Makino et al., Chem. Pharm. Bull. 33 (1984), 1195-1201. As said before,the size of the droplets can be controlled by changing theemulsification parameters, i.e. nature of emulsifier (more effective thesurfactant, i.e. the larger the hydrophilic to lipophilic balance, thesmaller the droplets) and the stirring conditions (faster and moreenergetic the agitation, the smaller the droplets).

In another variant, the interfacial wall forming polymer is dissolved inthe starting hydrophobic phase itself; the latter is emulsified intodroplets in the aqueous phase and the membrane around the droplets willform upon subsequent evaporation of this encapsulated hydrophobic phase.An example of this is reported by J. R. Farnand et al., PowderTechnology 22 (1978), 11-16 who emulsify a solution of polymer (e.g.polyethylene) in naphthalene in boiling water, then after cooling theyrecover the naphthalene in the form of a suspension of polymer boundedmicrobeads in cold water and, finally, they remove the naphthalene bysubjecting the microbeads to sublimation, whereby 25 μm microballoonsare produced. Other examples exist, in which a polymer is dissolved in amixed hydrophobic phase comprising a volatile hydrophobic organicsolvent and a water-soluble organic solvent, then this polymer solutionis emulsified in a water phase containing an emulsifier, whereby thewater-soluble solvent disperses into the water phase, thus aiding in theformation of the emulsion of microdroplets of the hydrophobic phase andcausing the polymer to precipitate at the interface; this is disclosedin EP-A-274.961 (H. Fessi).

The aforementioned techniques can be adapted to the preparation of airor gas filled microballoons suited for ultrasonic imaging provided thatappropriate conditions are found to control sphere size in the desiredranges, cell-wall permeability or imperviousness and replacement of theencapsulated liquid phase by air or a selected gas. Control of overallsphere size is obviously important to adapt the microballoons to usepurposes, i.e. injection or oral intake. The size conditions forinjection (about 0.5-10 μm average size) have been discussed previously.For oral application, the range can be much wider, being considered thatechogenicity increases with size; hence microballoons in several sizeranges between say 1 and 1000 μm can be used depending on the needs andprovided the membrane is elastic enough not to break during transit inthe stomach and intestine. Control of cell-wall permeability isimportant to ensure that infiltration by the injectable aqueous carrierphase is absent or slow enough not to impair the echographicmeasurements but, in cases, still substantial to ensure relatively fastafter-test biodegradability, i.e. ready metabolization of the suspensionby the organism. Also the microporous structure of the microballoonsenvelope (pores of a few nm to a few hundreds of nm or more formicroballoons envelopes of thickness ranging from 50-500 nm) is a factorof resiliency, i.e. the microspheres can readily accept pressurevariations without breaking. The preferred range of pore sizes is about50-2000 nm.

The conditions for achieving these results are met by using the methoddisclosed herein.

One factor which enables to control the permeability of themicroballoons membrane is the rate of evaporation of the hydrophobicphase relative to that of water in step (4) of the method e.g. underconditions of freeze drying. For instance if the evaporation in iscarried out between about -40° and 0° C., and hexane is used as thehydrophobic phase, polystyrene being the interfacially depositedpolymer, beads with relatively large pores are obtained; this is sobecause the vapour pressure of the hydrocarbon in the chosen temperaturerange is significantly greater than that of water, which means that thepressure difference between the inside and outside of the spheres willtend to increase the size of the pores in the spheres membrane throughwhich the inside material will be evaporated. In contrast, usingcyclooctane as the hydrophobic phase (at -17° C. the vapour pressure isthe same as that of water) will provide beads with very tiny poresbecause the difference of pressures between the inside and outside ofthe spheres during evaporation is minimized.

Depending on degree of porosity the microballoons of this invention canbe made stable in an aqueous carrier from several hours to severalmonths and give reproducible echographic signals for a long period oftime. Actually, depending on the polymer selected, the membrane of themicroballoons can be made substantially impervious when suspended incarrier liquids of appropriate osmotic properties, i.e. containingsolutes in appropriate concentrations. It should be noted that theexistence of micropores in the envelope of the microballoons of thepresent invention appears to be also related with the echographicresponse, i.e., all other factors being constant, microporous vesiclesprovide more efficient echographic signal than corresponding non-porousvesicles. The reason is not known but it can be postulated that when agas is in resonance in a closed structure, the damping properties of thelatter may be different if it is porous or non-porous.

Other non water soluble organic solvents which have a vapour pressure ofthe same order of magnitude between about -40° C. and 0° C. areconvenient as hydrophobic solvents in this invention. These includehydrocarbons such as for instance n-octane, cyclooctane, thedimethylcyclohexanes, ethyl-cyclohexane, hexane, 2-, 3- and4-methyl-heptane, 3-ethyl-hexane, toluene, xylene, 2-methyl-2-heptane,2,2,3,3-tetramethylbutane and the like. Esters such as propyl andisopropyl butyrate and isobutyrate, butyl-formate and the like, are alsoconvenient in this range. Another advantage of freeze drying is tooperate under reduced pressure of a gas instead of air, whereby gasfilled microballoons will result. Physiologically acceptable gases suchas CO₂, N₂ O, methane, Freon, helium and other rare gases are possible.Gases with radioactive tracer activity can be contemplated.

As the volatile solvent insoluble in water to be used for dissolving thepolymer to be precipitated interfacially, one can cite halo-compoundssuch as CCl₄, CH₃ Br, CH₂ Cl₂, chloroform, Freon, low boiling esterssuch as methyl, ethyl and propyl acetate as well as lower ethers andketones of low water solubility. When solvents not totally insoluble inwater are used, e.g. diethyl-ether, it is advantageous to use, as theaqueous phase, a water solution saturated with said solvent beforehand.

The aqueous phase in which the hydrophobic phase is emulslfied as anoil-in-water emulsion preferably contains 1-20% by weight ofwater-soluble hydrophilic compounds like sugars and polymers asstabilizers, e.g. polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP),polyethylene glycol (PEG), gelatin, polyglutamic acid, albumin, andpolysaccharides such as starch, dextran, agar, xanthan and the like.Similar aqueous phases can be used as the carrier liquid in which themicroballoons are suspended before use.

Part of this water-soluble polymer can remain in the envelope of themicroballoons or it can be removed by washing the beads beforesubjecting them to final evaporation of the encapsulated hydrophobiccore phase.

The emulsifiers to be used (0.1-5% by weight) to provide theoil-in-water emulsion of the hydrophobic phase in the aqueous phaseinclude most physiologically acceptable emulsifiers, for instance egglecithin or soya bean lecithin, or synthetic lecithins such as saturatedsynthetic lecithins, for example, dimyristoyl phosphatidyl choline,dipalmitoyl phosphatidyl choline or distearoyl phosphatidyl choline orunsaturated synthetic lecithins, such as dioleyl phosphatidyl choline ordilinoleyl phosphatidyl choline. Emulsifiers also include surfactantssuch as free fatty acids, esters of fatty acids with polyoxyalkylenecompounds like polyoxypropylene glycol and polyoxyethylene glycol;ethers of fatty alcohols with polyoxyalkylene glycols; esters of fattyacids with polyoxyalkylated sorbitan; soaps; glycerol-polyalkylenestearate; glycerol-polyoxyethylene ricinoleate; homo- and copolymers ofpolyalkylene glycols; polyethoxylated soya-oil and castor oil as well ashydrogenated derivatives; ethers and esters of sucrose or othercarbohydrates with fatty acids, fatty alcohols, these being optionallypolyoxyalkylated; mono-, di- and triglycerides of saturated orunsaturated fatty acids; glycerides or soya-oil and sucrose.

The polymer which constitutes the envelope or bounding membrane of theinjectable microballoons can be selected from most hydrophilic,biodegradable physiologically compatible polymers. Among such polymersone can cite polysaccharides of low water solubility, polylactides andpolyglycolides and their copolymers, copolymers of lactides and lactonessuch as ε-caprolactone, δ-valerolactone and, polypeptides. The greatversatility in the selection of synthetic polymers is another advantageof the present invention since, as with allergic patients, one may wishto avoid using microballoons made of natural proteins (albumin, gelatin)like in U.S. Pat. No. 4,276,885 or EP-A-324.938. Other suitable polymersinclude poly-(ortho)esters (see for instance U.S. Pat. No. 4,093,709;U.S. Pat. No. 4,131,648; U.S. Pat. No. 4,138,344; U.S. Pat. No.4,180,646); polylactic and polyglycolic acid and their copolymers, forinstance DEXON (see J. Heller, Biomaterials 1 (1980), 51;poly(DL-lactide-co-δ-caprolactone), poly(DL-lactide-co-δ-valerolactone),poly(DL-lactide-co-δ-butyrolactone), polyalkylcyanoacrylates;polyamides, polyhydroxy-butyrate; polydioxanone; poly-β-aminoketones(Polymer 23 (1982), 1693); polyphosphazenes (Science 193 (1976), 1214);and polyanhydrides. References on biodegradable polymers can be found inR. Langer et al., Macromol. Chem. Phys. C23 (1983), 61-126.Polyamino-acids such as polyglutamic and polyaspartic acids can also beused as well as their derivatives, i.e. partial esters with loveralcohols or glycols. One useful example of such polymers ispoly-(t.butyl-glutamate). Copolymers with other amino-acids such asmethionine, leucine, valine, proline, glycine, alamine, etc. are alsopossible. Recently some novel derivatives of polyglutamic andpolyaspartic acid with controlled biodegradability have been reported(see WO87/03891; U.S. Pat. No. 4,888,398 and EP-130.935 incorporatedhere by reference). These polymers (and copolymers with otheramino-acids) have formulae of the following type:

    --(NH--CHA--CO).sub.x (NH--CHX--CO).sub.y

where X designates the side chain of an amino-acid residue and A is agroup of formula --(CH₂)_(n) COOR¹ R² --OCOR (II), with R¹ and R² beingH or lover alkyls, and R being alkyl or aryl; or R and R¹ are connectedtogether by a substituted or unsubstituted linking member to provide 5-or 6- membered rings.

A can also represent groups of formulae:

    --(CH.sub.2).sub.n COO--CHR.sup.1 COOR                     (I)

and

    --(CH.sub.2).sub.n CO(NH--CHX--CO).sub.m NH--CH(COOH)--(CH.sub.2).sub.p COOH(III)

and corresponding anhydrides. In all these formulae n, m and p are lowerintegers (not exceeding 5) and x and y are also integers selected forhaving molecular weights not below 5000.

The aforementioned polymers are suitable for making the microballoonsaccording to the invention and, depending on the nature of substituentsR, R¹, R² and X, the properties of the membrane can be controlled, forinstance, strength, elasticity and biodegradability. For instance X canbe methyl (alanine), isopropyl (valine), isobutyl (leucine andisoleucine), benzyl (phenylalanine).

Additives can be incorporated into the polymer wall of the microballoonsto modify the physical properties such as dispersibility, elasticity andwater permeability. For incorporation in the polymer, the additives canbe dissolved in the polymer carrying phase, e.g. the hydrophobic phaseto be emulsified in the water phase, whereby they will co-precipitatewith the polymer during inter-facial membrane formation.

Among the useful additives, one may cite compounds which can"hydrophobize" the microballoons membrane in order to decrease waterpermeability, such as fats, waxes and high molecular-weighthydrocarbons. Additives which improve dispersibility of themicroballoons in the injectable liquid-carrier are amphipatic compoundslike the phospholipids; they also increase water permeability and rateof biodegradability.

Non-biodegradable polymers for making microballoons to be used in thedigestive tract can be selected from most water-insoluble,physiologically acceptable, bioresistant polymers including polyolefins(polystyrene), acrylic resins (polyacrylates, polyacrylonitrile),polyesters (polycarbonate), polyurethanes, polyurea and theircopolymers. ABS (acryl-butadiene-styrene) is a preferred copolymer.

Additives which increase membrane elasticity are the plasticizers likeisopropyl myristate and the like. Also, very useful additives areconstituted by polymers akin to that of the membrane itself but withrelatively low molecular weight. For instance when using copolymers ofpolylactic/polyglycolic type as the membrane forming material, theproperties of the membrane can be modified advantageously (enhancedsoftness and biodegradability) by incorporating, as additives, lowmolecular weight (1000 to 15,000 Dalton) polyglycolides or polylactides.Also polyethylene glycol of moderate to low M_(w) (e.g. PEG 2000) is auseful softening additive.

Preferably the plasticizers include isopropyl myristate, glycerylmonstearate and the like to control flexibility, the amphipaticsubstance include surfactants and phospholipids like the lecithins tocontrol permeability by increasing porosity while the hydrophobiccompounds include molecular weight hydrocarbon like the paraffin-waxesto reduce porosity.

The quantity of additives to be incorporated in the polymer forming theinter-facially deposited membrane of the present microballoons isextremely variable and depends on the needs. In some cases no additiveis used at all; in other cases amounts of additives which may reachabout 20% by weight of the polymer are possible.

The injectable microballoons of the present invention can be stored dryin the presence or in the absence of additives to improve conservationand prevent coalescence. As additives, one may select from 0.1 to 25% byweight of water-soluble physiologically acceptable compounds such asmannitol, galactose, lactose or sucrose or hydrophilic polymers likedextran, xanthan, agar, starch, PVP, polyglutamic acid, polyvinylalcohol(PVA), albumin and gelatin. The useful life-time of the microballoons inthe injectable liquid carrier phase, i.e. the period during which usefulechographic signals are observed, can be controlled to last from a fewminutes to several months depending on the needs; this can be done bycontrolling the porosity of the membrane from substantial imperviousnesstoward carrier liquids to porosities having pores of a few nanometers toseveral hundreds of nanometers. This degree of porosity can becontrolled, in addition to properly selecting the membrane formingpolymer and polymer additives, by adjusting the evaporation rate andtemperature in step (4) of the method of and properly selecting thenature of the compound (mixture of compounds) constituting thehydrophobic phase, i.e. the greater the differences in its partialpressure of evaporation with that of the water phase, the coarser thepores in the microballoons membrane will be. Of course, this control byselection of the hydrophobic phase can be further refined by the choiceof stabilizers and by adjusting the concentration thereof in order tocontrol the rate of water evaporation during the forming of themicroballoons. All these changes can easily be made by skilled personswithout exercizing inventiveness and need not be further discussed.

It should be remarked that although the microballoons of this inventioncan be marketed in the dry state, more particularly when they aredesigned with a limited life time after injection, it may be desirableto also sell ready preparations, i.e. suspensions of microballoons in anaqueous liquid carrier ready for injection or oral administration. Thisrequires that the membrane of the microballoons be substantiallyimpervious (at least for several months or more) to the carrier liquid.It has been shown in this description that such conditions can be easilyachieved with the present method by properly selecting the nature of thepolymer and the interfacial deposition parameters. Actually parametershave been found (for instance using the polyglutamic polymer (where A isthe group of formula II) and cyclooctane as the hydrophobic phase) suchthat the porosity of the membrane after evaporation of the hydrophobicphase is so tenuous that the microballoons are substantially imperviousto the aqueous carrier liquid in which they are suspended.

A preferred administrable preparation for diagnostic purposes comprisesa suspension in buffered or unbuffered saline (0.9% aqueous NaCl; buffer10 mM tris-HCl) containing 10⁸ -10¹⁰ vesicles/ml. This can be preparedmainly according to the directions of the Examples below, preferablyExamples 3 and 4, using poly-(DL-lactide) polymers from the CompanyBoehringer, Ingelheim, Germany.

The following Examples illustrate the invention practically.

EXAMPLES 1

One gram of polystyrene was dissolved in 19 g of liquid naphthalene at100° C. This naphthalene solution was emulsified at 90°-95° C. into 200ml of a water solution of polyvinyl alcohol (PVA) (4% by weight)containing 0.1% of Tween-40 emulsifier. The emulsifying head was aPolytron PT-3000 at about 10,000 rpm.

Then the emulsion was diluted under agitation with 500 ml of the sameaqueous phase at 15° C. whereby the naphthalene droplets solidified intobeads of less than 50 μm as ascertained by passing through a 50 μm meshscreen. The suspension was centrifugated under 1000 g and the beads werewashed with water and recentrifugated. This step was repeated twice.

The beads were resuspended in 100 ml of water with 0.8 g of dissolvedlactose and the suspension was frozen into a block at 31 30° C. Theblock was thereafter evaporated under about 0.5-2 Torr between about -20and -10° C. Air filled microballoons of average size 5-10 μm andcontrolled porosity were thus obtained which gave an echographic signalat 2.25 and 7.5 MHz after being dispersed in water (3% dispersion byweight). The stability of the microballoons in the dry state waseffective for an indefinite period of time: once suspended in an aqueouscarrier liquid the useful life-time for echography was about 30 min ormore. Polystyrene being non-biodegradable, this material was not favoredfor injection echography but was useful for digestive tractinvestigations. This Example clearly establishes the feasibility of themethod of the invention.

EXAMPLES 2

A 50:50 copolymer mixture (0.3 g) of DL-lactide and glycolide (Du PontMedisorb) and 16 mg of egg-lecithin were dissolved in 7.5 ml of CHCl₃ togive solution (1).

A solution (2) containing 20 mg of paraffin-wax (N.P. 54°-56° C.) in 10ml of cyclooctane (M.P. 10°-13°) was prepared and emulsified in 150 mlof a water solution (0.13% by weight) of Pluronic F-108 (a blockcopolymer of ethylene oxide and propylene oxide) containing also 1.2 gof CHCl₃. Emulsification was carried out at room temperature for 1 minwith a Polytron head at 7000 rpm. Then solution (1) was added underagitation (7000 rpm) and, after about 30-60 sec, the emulsifier head wasreplaced by a helical agitator (500 rpm) and stirring was continued forabout 3 hrs at room temperature (22° C.). The suspension was passedthrough a 50 μm screen and frozen to a block which was subsequentlyevaporated between -20° and 0° C. under high-vacuum (catching trap -60°C. to -80° C.). There were thus obtained 0.264 g (88%) of air-filledmicroballoons stable in the dry state.

Suspensions of said microballoons in water (no stabilizers) gave astrong echographic signal for at least one hour. After injection in theorganism, they biodegraded in a few days.

EXAMPLES 3

A solution was made using 200 ml of tetrahydrofuran (THF), 0.8 g of a50:50 DL-lactide/glycolide copolymer (Boehringer AG), 80 mg ofegg-lecithin, 64 mg of paraffin-wax and 4 ml of octane. This solutionwas emulsified by adding slowly into 400 ml of a 0.1 aqueous solution ofPluronic F-108 under helical agitation (500 r.p.m.). After stirring for15 min, the milky dispersion was evaporated under 10-12 Torr 25° C. in arotavapor until its volume was reduced to about 400 ml. The dispersionwas sieved on a 50 μm grating, then it was frozen to -40° C. andfreeze-dried under about 1 Torr. The residue, 1.32 g of very finepowder, was taken with 40 ml of distilled water which provided, after 3min of manual agitation, a very homogeneous dispersion of microballoonsof average size 4.5 μm as measured using a particle analyzer(Mastersizer from Malvern). The concentration of microballoons (CoulterCounter) was about 2×10⁹ /ml. This suspension gave strong echographicsignals which persisted for about 1 hr.

If in the present example, the additives to the membrane polymer areomitted, i.e. there is used only 800 mg of the lactide/glycolidecopolymer in the THF/octane solution, a dramatic decrease in cell-wallpermeability is observed, the echographic signal of the dispersion inthe aqueous carrier not being significantly attenuated after 3 days.

Using intermediate quantities of additives provided beads withcontrolled intermediate porosity and life-time.

EXAMPLES 4

There was used in this Example a polymer of formula defined in claim 8in which the side group has formula (II) where R¹ and R² are hydrogenand R is tert.butyl. The preparation of this polymer (defined aspoly-POMEG) is described in U.S. Pat. No. 4,888,398.

The procedure was like in Example 3, using 0.1 g poly-POMEG, 70 ml ofTHF, 1 ml of cyclooctane and 100 ml of a 0.1% aqueous solution ofPluronic F-108. No lecithin or high-molecular weight hydrocarbon wasadded. The milky emulsion was evaporated at 27° C./10 Torr until theresidue was about 100 ml, then it was screened on a 50 μm mesh andfrozen. Evaporation of the frozen block was carried out (0.5-1 Torr)until dry. The yield was 0.18 g because of the presence of thesurfactant. This was dispersed in 10 ml of distilled water and countedwith a Coulter Counter. The measured concentration was found to be1.43×10⁹ microcapsules/ml, average size 5.21 μm as determined with aparticle analyzer (Mastersizer from Malvern). The dispersion was diluted100×, i.e. to give about 1.5×10⁷ microspheres/ml and measured forechogenicity. The amplitude of the echo signal was 5 times greater at7.5 MHz than at 2.25 MHz. These signals were reproducible for a longperiod of time.

Echogenicity measurements were performed with a pulse-echo systemconsisting of a plexiglas specimen holder (diameter 30 mm) with a 20 μmthick Mylar acoustic window, a transducer holder immersed in a constanttemperature water bath, a pulser-receiver (Accutron M3010JS) with anexternal pre-amplifier with a fixed gain of 40 dB and an internalamplifier with gain adjustable from -40 to +40 dB and interchangeable 13mm unfocused transducers. A 10 MHz low-pass filter was inserted in thereceiving part to improve the signal to noise ratio. The A/D board inthe IBM PC was a Sonotek STR 832. Measurements were carried out at 2.25,3.5, 5 and 7.5 MHz.

If in the present Example, the polymer used is replaced bylactic-lactone copolymers, the lactones being δ-butyrolactone,δ-valerolactone or ε-caprolactone (see Fukuzaki et al., J. BiomedicalMater. Res. 25 (1991), 315-328), similar favorable results wereobtained. Also in a similar context, polyalkylcyano-acrylates andparticularly a 90:10 copolymer poly(DL-lactide-co-glycolide) gavesatisfactory results. Finally, a preferred polymer is a poly(DL-lactide)from the Company Boehringer-Ingelheim sold under the name "ResomerR-206" or Resomer R-207.

EXAMPLES 5

Two-dimensional echocardiography was performed using an Acuson-128apparatus with the preparation of Example 4 (1.43×10^(9/) ml) in anexperimental dog following peripheral vein injection of 0.1-2 ml of thedispersion. After normally expected contrast enhancement imaging of theright heart, intense and persistent signal enhancement of the left heartwith clear outlining of the endocardium was observed, thereby confirmingthat the microballoons made with poly-POMEG (or at least a significantpart of them) were able to cross the pulmonary capillary circulation andto remain in the blood-stream for a time sufficient to perform efficientechographic analysis.

In another series of experiments, persistent enhancement of the Dopplersignal from systemic arteries and the portal vein was observed in therabbit and in the rat following peripheral vein injection of 0.5-2 ml ofa preparation of microballoons prepared as disclosed in Example 4 butusing poly(DL-lactic acid) as the polymer phase. The composition usedcontained 1.9×10⁸ vesicles/ml.

Another composition prepared also according to the directions of Example4 was achieved using poly(tert.butylglutamate). This composition (0.5ml) at dilution of 3.4×10⁸ microballoons/ml was injected in the portalvein of rats and gave persistent contrast enhancement of the liverparenchyma.

EXAMPLES 6

A microballoon suspension (1.1×10⁹ vesicles/ml) was prepared asdisclosed in Example 1 (resin=polystyrene). One ml of this suspensionwas diluted with 100 ml of 300 mM mannitol solution and 7 ml of theresulting dilution was administered intragastrically to a laboratoryrat. The animal was examined with an Acuson-128 apparatus for2-dimensional echography imaging of the digestive tract which clearlyshowed the single loops of the small intestine and of the colon.

We claim:
 1. An ultrasonic contrast agent comprising a suspension of airor gas microballoons in a physiologically acceptable aqueous carrierliquid in a concentration of 10⁵ to 10¹⁰ microballoons/ml, themicroballoons having a mean size in the range of 0.5 to 1,000 microns,are bounded by a soft, elastic, 50-500 nm thick polymer membrane filledwith air or a physiologically acceptable gas, the membrane beingtemporarily deformable under pressure variations and made from abiodegradable, synthetic, resilient, interfacially depositable polymerselected from the group consisting of polysaccharides, polylactides,polyglycolides, copolymers of polylactides and polyglycolides,copolymers of lactides and lactones, poly-(ortho)esters, polydioxanone,poly-β-aminoketones, polyphosphazenes, polyanhydrides andpolyalkyl-(cyano)acrylates.
 2. The ultrasonic contrast agent of claim 1,wherein the microballoons have size in the range of 0.5-10 μm and themembrane is either impervious or permeable to bioactive liquids.
 3. Theultrasonic contrast agent of claim 1, wherein the polymer membrane isporous and has porosity ranging from 50 to 2000 nanometers.
 4. Theultrasonic contrast agent of claim 1, wherein the membrane polymer ispolyglutanic or polyaspartic acid esters or amides and their copolymerswith amino acids.
 5. The ultrasonic contrast agent of claim 4, whereinthe polyglutamic and aspartic acid esters or amides have side functionshaving formulae

    (CH.sub.2).sub.n COO--CHR.sup.1 COOR                       (I),

or

    (CH.sub.2).sub.n COOCR.sup.1 R.sup.2 --O--COR              (II)

or

    (CH.sub.2).sub.n CO(NH--CHX--CO).sub.m NHCH(COOH)--(CH.sub.2).sub.p COOH(III),

wherein R is an alkyl or aryl substituent; R¹ and R² are H or loweralkyls, or R and R¹ are connected together by a substituted orunsubstituted linking member to form a 5- or 6-membered ring; n is 1 or2; p is 1, 2 or 3; m is an integer from 1 to 5 and X is a side chain ofan amino acid residue.
 6. The ultrasonic contrast agent of claim 1,wherein the polymer membrane also includes a plasticizer selected fromisopropyl myristate and glyceryl monostearate.
 7. The ultrasoniccontrast agent of claim 1, wherein the polymer membrane also includes asurfactant.
 8. The ultrasonic contrast agent of claim 1, wherein thepolymer membrane also includes a phospholipid.
 9. The ultrasoniccontrast agent of claim 1, wherein the polymer membrane also includes aparaffin wax.
 10. The ultrasonic contrast agent of claim 1, whereinmicroballoons are present in the suspension in a concentration of about10₆ to 10₁₀ microballoons per ml.
 11. The ultrasonic contrast agent ofclaim 10, wherein microballoons are present in the suspension in aconcentration of about 10⁸ to 10¹⁰ microballoons per ml.
 12. Theultrasonic contrast agent of claim 1, wherein the physiologicallyacceptable gas in the microballoons is selected from the groupconsisting of CO₂, N₂ O, methane, helium and a freon.
 13. The ultrasoniccontrast agent of claim 1, wherein the physiologically acceptable gas inthe microballoons is a fluorine containing gas.